Ergosterol is the principal membrane sterol of fungi. It is structurally similar to its animal counterpart, cholesterol, except that ergosterol has a methyl group and two double bonds not present in cholesterol. In yeast, ergosterol affects membrane fluidity and permeability and plays an essential role in the yeast cell cycle. Yeast cells can take up cholesterol and decrease their requirement for ergosterol to very low levels, but cholesterol alone cannot completely substitute for ergosterol (Gaber, R. F., et al., Mol. Cell. Biol. 9:3447-3456 (1989)).
Though the biosynthesis of ergosterol in fungi involves steps distinct from cholesterol biosynthesis in animals, sterol biosynthesis in different organisms share many common steps. Implicated in sterol biosynthesis is at least one cytochrome P450. The term "cytochrome P450" is a trivial name for a class of cytochromes that includes a number of heme proteins exhibiting a characteristic absorption maximum at 450 nm when combined with CO in the reduced state (`P` denotes pigment; hence, the name). These cytochromes occur in most animal tissues, plants and microorganisms and catalyze the monooxygenation of a vast variety of hydrophobic substances, including lipophilic endogenous compounds and xenobiotics, serving as oxygenating catalysts in the presence of one or more electrontransfer proteins or redox enzymes.
Several distinct cytochrome P450 proteins have been described in yeast and fungi, and the structural genes encoding these proteins, have been isolated (Kalb, V. F., et al., Gene 45:237-245 (1986) and Turi, T. G., and Loper, J. C., J. Biol. Chem. 267:2046-2056 (1992)). Of these, the most thoroughly characterized is the lanosterol 14-.alpha.-demethylase of Saccharomyces cerevisiae (ERG11), which was one of the first S. cerevisiae enzymes in the ergosterol biosynthetic pathway to be cloned. The sequence of the gene has been published, and the protein shown to be essential for aerobic growth (Kalb, V., et al., DNA 6: 529-537 (1987)). This cytochrome P450 catalyzes the oxidative removal of a methyl group at the carbon at position 14 of lanosterol during the biosynthesis of ergosterol; the mammalian orthologue catalyzes the identical reaction in cholesterol biosynthesis. In addition, lanosterol 14-.alpha.-demethylase is the specific target of a number of antifungal agents such as ketoconazole, miconazole, econazole, dinaconazole and itraconazole.
Multiple regulatory elements control the expression of the ERG11 gene encoded in Saccharomyces cerevisiae (Turi and Loper, cited above). Message levels for the gene increase during yeast growth on glucose, in the presence of heme, during oxygen-limiting conditions, and during anaerobic growth. Genetic analysis indicates there are multiple upstream activating and repressor sequences in the ERG11 promoter (ibid.). At least one of the upstream activating sequences can be activated by other proteins, and repression depends on other repressors. In addition, gene expression appears to be coordinated with the expression of another gene, the CPR1 gene for NADPH-cytochrome P450 reductase, which codes for a flavoprotein that serves as an electron transfer enzyme from NADPH to cytochrome P450 and thus acts in concert with it (ibid.). Complicating this complex regulation system are the mechanisms controlling overall gene regulation in yeast (reviewed by Hinnebusch, A. G., Micro. Rev. 52:248-273 (1988)).